Nucleic acids encoding for non-infectious, replication-defective, self-assembling HIV-1 viral particles containing antigenic markers in the gag coding region

Abstract

Non-infectious, retrovirus-like particles comprise an assembly of an env gene product, a pol gene product and a gag gene product contain an antigenic marker which is non-retroviral or non-HIV retroviral. In one embodiment, the marker comprises an amino acid sequence containing an epitope inserted into the gag gene product at an antigenically-active insertion site. In another embodiment, the marker comprises an antigenic anchor sequence operatively connected to the env gene product replacing endogenous anchoring function. The corresponding nucleic acid molecules are described. The non-infectious, retrovirus-like particles have utility in in vivo administration including to humans and in diagnosis. The presence of the antigenic marker enables recognition that antiserum containing anti-retroviral antibodies has been generated by exposure to the non-infectious retrovirus-like particles by testing for antibodies specific to the antigenic marker.

What we claim is: 
     
       1. A nucleic acid molecule encoding a non-infectious, replication-deficient, retrovirus-like particle containing a heterologous antigenic marker, comprising: a modified HIV genome, deficient in long terminal repeats (LTRs), containing gag, pol and env genes in their natural genomic arrangement and a heterologous nucleic acid insert encoding at least one non-retroviral, non-mammalian antigenic marker wherein said marker, when presented in the context of the retrovirus-like particle, capable of generating an immune response specific to said marker when the encoded particle is administered to a host.   
     
     
       2. The nucleic acid molecule of claim 1, wherein the segment encoding the at least one antigenic marker contains between 15 and 300 nucleotides. 
     
     
       3. The nucleic acid molecule of claim 2, wherein the segment encoding the at least one antigenic marker contains between 30 to 225 nucleotides. 
     
     
       4. The nucleic acid molecule of claim 2, wherein the segment encoding the at least one antigenic marker encodes at least one antigenic epitope from tobacco mosaic virus coat protein. 
     
     
       5. The nucleic acid molecule of claim 4 wherein the at least one antigenic epitope from tobacco mosaic virus coat protein includes an amino acid sequence AFDTRNRIIEVEN (SEQ ID NO: 1) or a portion, variation or mutant thereof capable of eliciting antibodies that recognize the sequence AFDTRNRIIEVEN (SEQ ID NO:1). 
     
     
       6. The nucleic acid molecule of claim 1, wherein the segment encoding the at least one antigenic marker is contained within the gag gene to provide a modified gag gene encoding a hybrid war gene product having the particle-forming characteristics of unmodified gag gene product. 
     
     
       7. The nucleic acid molecule of claim 6, wherein the segment encoding the at least one antigenic marker is inserted into the gag gene to provide the antigenic marker at an antigenically-active insertion site in the hybrid gag gene product. 
     
     
       8. The nucleic acid molecule of claim 7 wherein the segment encoding the at least one antigenic marker is inserted at an insertion site, located at the PstI site at nucleotide 1415 of the gag gene of HIV-1 LAI isolate or the corresponding location of other retroviral gag genes. 
     
     
       9. A nucleic acid molecule encoding a non-infectious, replication-deficient, HIV retrovirus-like particle containing a heterologous antigenic marker, comprising: a modified HIV retroviral genome deficient in long terminal repeats (LTRs) and containing gag, pol and env genes in their natural genomic arrangement and a heterologous nucleic acid insert encoding at least one non-retroviral non-mammalian antigenic marker wherein said modified genome comprises from one to four copies of a nucleic acid sequence selected from the group consisting of:   (a) 5' GCATTCGACACTAGAAATAGAATAATAGAAGTTGAAAAT 3' (SEQ ID NO: 5); or   (b) 3'CGTAAGCTGTGATCTTTATCTTATTATCTTCAACTTTTA5' (SEQ. ID NO: 6);, and said marker is inserted into the PstI site at nucleotide 1415 of the HIV-1 LAI  gag gene, which when presented in the context of the retrovirus-like particles, is capable of encoded particle is administered to a host.   
     
     
       10. An immunogenic composition capable of eliciting a retroviral specific immune response and a specific immune response against a non-retroviral marker, comprising the nucleic acid molecule of claim 1 and a carrier therefor. 
     
     
       11. The immunogenic composition of claim 10 formulated for mucosal or parenteral administration. 
     
     
       12. The immunogenic composition of claim 10 formulated for oral, anal, vaginal, or intranasal administration. 
     
     
       13. The immunogenic composition of claim 11 further comprising at least one other immunogenic or immunostimulating material. 
     
     
       14. The composition of claim 13 wherein the at least one other immunostimulating material is an adjuvant. 
     
     
       15. The composition of claim 14, wherein the adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, Freund's incomplete adjuvant, and QS21. 
     
     
       16. A method of immunizing a host to produce a retroviral specific immune response and a specific immune response against the antigenic marker, comprising administering to the host an immunoeffective amount of the immunogenic composition of claim 10. 
     
     
       17. A method of identifying antiserum generated by immunization with the immunogenic composition of claim 10, comprising: detecting antibodies specific for said antigenic marker in said antiserum.